PROJECT SUMMARY Craniofacial abnormalities are largely attributed to defects in the formation, migration and differentiation of cranial neural crest cells (CNCCs). Since CNCCs are developmentally plastic, understanding the genetic and molecular pathways controlling the specification of chondrocyte and osteoblast lineages from CNCC is a prerequisite for interpreting the etiology of craniofacial skeletal disorders. After the discovery of bone morphogenetic proteins (BMPs), many elegant studies have revealed their significant role in skeletal development. However, it is still unclear why BMPs are capable of changing the fate and lineage of undifferentiated stem cells of CNCCs toward skeletogenic cells. Especially, despite the importance of BMPs during craniofacial bone development, it is poorly understood how BMP signaling in CNCCs regulates craniofacial cartilage formation. In our preliminary studies, we employed the Cre-LoxP system that controls BMP signaling in a BMP receptor-specific manner in the mouse. It has been reported that gain-of-function mutations in components of the BMP axis cause craniofacial abnormalities in humans. Consistent with this observation, we previously reported that augmentation of BMP signaling in CNCCs through BMPR1A, one of the BMP type I receptors, causes craniosynostosis due to ectopic cartilage formation in cranial sutures. In this proposal, we focus on another BMP type I receptor, ACVR1. Embryos with augmented BMP signaling through ACVR1 in CNCCs (?ACVR1 mutants? hereafter) displayed jaw malformation and cleft lip, which are distinct craniofacial phenotypes from those of BMPR1A mutants. Interestingly, ACVR1 mutants displayed the enhanced cartilage growth in the face with upregulation of Sox9, a key transcription factor for chondrogenesis. Preliminary screenings revealed that the levels of mammalian target of rapamycin (mTOR) were significantly elevated in ACVR1 mutants. Importantly, inhibition of mTOR signaling by rapamycin rescued the craniofacial cartilage malformation in ACVR1 mutants, indicating that mTOR signaling triggered by the augmentation of BMP is responsible for the enhanced endochondral ossification. Of note, primary cilia, which are microtubule-based antenna-like organelles, were enriched in CNCC-derived chondrocytes in ACVR1 mutants, and the suppression of a ciliary protein rescued the craniofacial cartilage abnormalities in ACVR1 mutants. These results suggest that primary cilia in ACVR1 mutants are responsible for the etiology of enhanced cartilage growth in the face. Our central hypothesis here is that BMP signaling through ACVR1 in CNCCs regulates mTOR, which is required for primary cilium formation during craniofacial cartilage development. We will test our hypothesis by pursuing the specific aims (1) To examine how BMP signaling in CNCCs regulates primary cilium formation during chondrogenesis, and (2) To examine how BMP-mTOR-cilia axis governs chondrogenesis during facial development. Our study will uncover the molecular details of how the novel axis of BMP-mTOR-primary cilia in CNCCs is critical for craniofacial cartilage formation.